Chapter

1. You wish to perform a resolution of your restriction enzyme-digested DNA fragments. The size of the expected products ranges 500-100 bp. You discover two agarose gels polymerizing on the bench. One is 5% agarose. The other is 2% agarose. Which one might you use to resolve your fragments?

2. After completion of the run of fragments along with the proper molecular weight standard on the agarose gel, suppose a. or b. was observed. What might be explanations for these? (Assume you have included a molecular weight marker in your run.)

a. The gel is blank (no bands, no molecular weight standard).

Samples were not loaded properly. Electrodes were switched. Gel was not stained properly.

b. Only the molecular weight standard is visible.

Samples were not loaded properly.

Samples were diluted, degraded, or otherwise compromised.

Gel was not stained evenly.

3. How does PFGE separate larger fragments more efficiently than standard electrophoresis?

Repeated reorientation forces larger fragments through the gel matrix more effectively.

4. A 6% solution of 19:1 acrylamide is mixed, de-aerated, and poured between glass plates for gel formation. After an hour, the solution is still liquid. What might be one explanation for the gel not polymerizing?

No catalyst was added.

5. A gel separation of RNA yields aberrantly migrating bands and smears. Suggest two possible explanations for this observation.

Poor RNA sample quality.

Inadequate denaturation of the RNA before loading.

6. Why does DNA not resolve well in solution (without a gel matrix)?

The size and charge of DNA have opposing effects on migration.

7. Why is SyBr green less toxic than EtBr?

SyBr green binds in the minor groove of DNA, unlike EtBr, which intercalates between the bases. Intercalation by EtBr is more likely to cause DNA mutations.

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